Links

Research & Scholarship

Current Research and Scholarly Interests

We are studying how the location and identity of presynaptic calcium channels is regulated. Voltage-gated Ca2+ channels provide the critical link between the firing of a presynaptic nerve terminal and its release of neurotransmitter. The Ca2+ channels must be positioned very close to sites of vesicle fusion, and come in diverse forms with distinct activity-dependence, responsiveness to GABA, dopamine, acetylcholine and other neuromodulators, and susceptibility to neurological disorders such as migraine, ataxia or dystonia. Our working hypothesis involves molecular slots for particular types of channels. Slots regulate the mix of channel types and also help explain how defective channels might displace normal ones in genetically dominant disorders.

Our lab is particularly interested in studying multiple modes of synaptic vesicle fusion. The opening of Ca2+ channels drives at least two distinct forms of fusion. In the classical mode, the vesicle membrane fully merges with and flattens into the presynaptic membrane (full collapse fusion). In a newly characterized mode, termed kiss-and-run the connection between the vesicle interior and the external medium lasts long enough to allow passage of neurotransmitter, but the connection is severed before the identity of the vesicle is lost. We study the dynamic properties and functional implications of both fusion modes by loading single synaptic vesicles with single photoluminescent reporter particlesquantum dots. Sharp distinctions between full collapse fusion and kiss-and-run are now in hand. Experiments are underway to monitor the same fusion event optically and electrophysiologically.

One area of intense attention in our lab is the fundamental unit of cell-cell communication between brain neurons: quantal synaptic transmission. Presynaptic release of a packet of neurotransmitter, for example, glutamate, causes activation of postsynaptic receptors and a brief flow of current that promotes firing of the postsynaptic cell. We work on neuronal mechanisms that allow synapses to adapt to a sudden or long-lasting change in their level of activity. For example, blockade of impulses or of postsynaptic glutamate receptors causes a cascade of biochemical events that eventually leads to readjustment of critical molecular players on both sides of the synapse. We use state-of-the art methods to pin down the cell biology of changes in synaptic strength, of importance for adaptation of brain networks in learning and memory. Ongoing work in cultures of isolated neurons and brain slices

We study how synaptic transmission and depolarization cause changes in neuronal gene expression. Despite its importance, signaling from synapse or surface membrane to nucleus is only partly understood. One example of such signaling involves a local increase in Ca2+ concentration near a class of Ca2+ channels (L-type) different from those that trigger presynaptic transmitter release, subsequently leading to activation of an exemplar transcription factor, CREB, a regulator of transcription of many important neuronal genes. Our approach is to combine physiological approaches (how fast, how steeply voltage-dependent, how is signal transduced) and biochemical experiments using cDNA microarrays (which genes, in what context, what relationship to learning and memory).

Abstract

Down syndrome (DS) is the most prevalent form of mental retardation caused by genetic abnormalities in humans. This has been successfully modeled in mice to generate the Ts65Dn mouse, a genetic model of DS. This transgenic mouse model shares a number of physical and functional abnormalities with people with DS, including changes in the structure and function of neuronal circuits. Significant abnormalities in noradrenergic (NE-ergic) afferents from the locus coeruleus to the hippocampus, as well as deficits in NE-ergic neurotransmission are detected in these animals. In the current study we characterized in detail the behavioral phenotype of Ts65Dn mice, in addition to using pharmacological tools for identification of target receptors mediating the learning and memory deficits observed in this model of DS. We undertook a comprehensive approach to mouse phenotyping using a battery of standard and novel tests encompassing: (i) locomotion (Activity Chamber, PhenoTyper, and CatWalk), (ii) learning and memory (spontaneous alternation, delayed matching-to-place water maze, fear conditioning, and Intellicage), and (iii) social behavior. Ts65Dn mice showed increased locomotor activity in novel and home cage environments. There were significant and reproducible deficits in learning and memory tests including spontaneous alternation, delayed matching-to-place water maze, Intellicage place avoidance and contextual fear conditioning. Although Ts65Dn mice showed no deficit in sociability in the 3-chamber test, a marked impairment in social memory was detected. Xamoterol, a β1-adrenergic receptor (β1-ADR) agonist, effectively restored the memory deficit in contextual fear conditioning, spontaneous alternation and novel object recognition. These behavioral improvements were reversed by betaxolol, a selective β1-ADR antagonist. In conclusion, our results demonstrate that this mouse model of Down syndrome displays cognitive deficits which are mediated by an imbalance in the noradrenergic system. In this experimental model of Down syndrome a selective activation of β1-ADR does restore some of these behavioral deficits. Further mechanistic studies will be needed to investigate the failure of noradrenergic system and the role of β1-ADR in cognitive deficit and pathogenesis of DS in people. Restoring NE neurotransmission or a selective activation of β1)-ADR needs to be further investigated for the development of any potential therapeutic strategy for symptomatic relief of memory deficit in DS. Furthermore, due to the significant involvement of noradrenergic system in the cardiovascular function further safety and translational studies will be needed to ensure the safety and efficacy of this approach.

Abstract

In excitable cells, membrane depolarization and activation of voltage-gated Ca²+ (Ca(V)) channels trigger numerous cellular responses, including muscle contraction, secretion, and gene expression. Yet, while the mechanisms underlying excitation-contraction and excitation-secretion coupling have been extensively characterized, how neuronal activity is coupled to gene expression has remained more elusive. In this article, we will discuss recent progress toward understanding the relationship between patterns of channel activity driven by membrane depolarization and activation of the nuclear transcription factor CREB. We show that signaling strength is steeply dependent on membrane depolarization and is more sensitive to the open probability of Ca(V) channels than the Ca²+ entry itself. Furthermore, our data indicate that by decoding Ca(V) channel activity, CaMKII (a Ca²+/calmodulin-dependent protein kinase) links membrane excitation to activation of CREB in the nucleus. Together, these results revealed some interesting and unexpected similarities between excitation-transcription coupling and other forms of excitation-response coupling.

Abstract

Prolonged AMPA-receptor blockade in hippocampal neuron cultures leads to both an increased expression of GluA1 postsynaptically and an increase in vesicle pool size and turnover rate presynaptically, adaptive changes that extend beyond simple synaptic scaling. As a molecular correlate, expression of the β Ca(2+)/CaM-dependent kinase type II (βCaMKII) is increased in response to synaptic inactivity. Here we set out to clarify the role of βCaMKII in the various manifestations of adaptation. Knockdown of βCaMKII by lentiviral-mediated expression of shRNA prevented the synaptic inactivity-induced increase in GluA1, as did treatment with the CaM kinase inhibitor KN-93, but not the inactive analog KN-92. These results demonstrate that, spurred by AMPA-receptor blockade, up-regulation of βCaMKII promotes increased GluA1 expression. Indeed, transfection of βCaMKII, but not a kinase-dead mutant, increased GluA1 expression on dendrites and elevated vesicle turnover (Syt-Ab uptake), mimicking the effect of synaptic inactivity on both sides of the synapse. In cells with elevated βCaMKII, relief of synaptic-activity blockade uncovered an increase in the frequency of miniature excitatory postsynaptic currents that could be rapidly and fully suppressed by PhTx blockade of GluA1 receptors. This increased mini frequency involved a genuine presynaptic enhancement, not merely an increased abundance of synapses. This finding suggests that Ca(2+) flux through GluA1 receptors may trigger the acute release of a retrograde messenger. Taken together, our results indicate that synaptic inactivity-induced increases in βCaMKII expression set in motion a series of events that culminate in coordinated pre- and postsynaptic adaptations in synaptic transmission.

Abstract

Prolonged blockade of AMPA-type glutamate receptors in hippocampal neuron cultures leads to homeostatic enhancements of pre- and postsynaptic function that appear correlated at individual synapses, suggesting some form of transsynaptic coordination. The respective modifications are important for overall synaptic strength but their interrelationship, dynamics, and molecular underpinnings are unclear. Here we demonstrate that adaptation begins postsynaptically but is ultimately communicated to presynaptic terminals and expressed as an accelerated turnover of synaptic vesicles. Critical postsynaptic modifications occur over hours, but enable retrograde communication within minutes once AMPA receptor (AMPAR) blockade is removed, causing elevation of both spontaneous and evoked vesicle fusion. The retrograde signaling does not require spiking activity and can be interrupted by NBQX, philanthotoxin, postsynaptic BAPTA, or external sequestration of BDNF, consistent with the acute release of retrograde messenger, triggered by postsynaptic Ca(2+) elevation via Ca(2+)-permeable AMPARs.

Abstract

Miniature synaptic events, resulting from spontaneous presynaptic release of neurotransmitter in the absence of an action potential, are often used to assay neural connectivity and are thought to play a pivotal role in the development and maintenance of neuronal circuits. In this issue of Neuron, Trigo et al. identify a new class of miniature synaptic event, called "preminis," that originate from and are subsequently detected by the presynaptic terminals of GABAergic neurons in the molecular layer of cerebellum. Remarkably, these events easily outnumber conventional minis. Their restriction to a relatively narrow time window (<15 days after birth) is a clue that they may play a critical role in wiring up interneurons within the developing cerebellar circuitry.

Abstract

Synaptic transmission at CNS synapses is often mediated by joint actions of multiple Ca(2+) channel subtypes, most prominently, P/Q- and N-type. We have proposed that P/Q-type Ca(2+) channels saturate type-preferring slots at presynaptic terminals, which impose a ceiling on the synaptic efficacy of the channels. To test for analogous interactions for presynaptic N-type Ca(2+) channels, we overexpressed their pore-forming Ca(V)2.2 subunit in cultured mouse hippocampal neurons, recorded excitatory synaptic transmission from transfected cells, and dissected the contributions of N-, P/Q-, and R-type channels with subtype-specific blockers. Overexpression of Ca(V)2.2 did not increase the absolute size of the EPSC even though somatic N-type current was augmented by severalfold. Thus, the strength of neurotransmission is saturated with regard to levels of Ca(2+) channel expression for both N-type and P/Q-type channels. Overexpression of Ca(2+)-impermeable Ca(V)2.2 subunits decreased EPSC size, corroborating competition for channel slots. Striking asymmetries between N- and P/Q-type channels emerged when their relative contributions were compared with channel overexpression. Overexpressed N-type channels could competitively displace P/Q-type channels from P/Q-preferring slots and take over the role of supporting transmission. The converse was not found with overexpression of P/Q-type channels, regardless of their C-terminal domain. We interpret these findings in terms of two different kinds of presynaptic slots at excitatory synapses, one accepting N-type channels but rejecting P/Q-type (N(specific)) and the other preferring P/Q-type but also accepting N-type (PQ(preferring)). The interaction between channels and slots governs the respective contributions of multiple channel types to neurotransmission and, in turn, the ability of transmission to respond to various stimulus patterns and neuromodulators.

Inhibitory role for GABA in autoimmune inflammationPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABhat, R., Axtell, R., Mitra, A., Miranda, M., Lock, C., Tsien, R. W., Steinman, L.2010; 107 (6): 2580-2585

Abstract

GABA, the principal inhibitory neurotransmitter in the adult brain, has a parallel inhibitory role in the immune system. We demonstrate that immune cells synthesize GABA and have the machinery for GABA catabolism. Antigen-presenting cells (APCs) express functional GABA receptors and respond electrophysiologically to GABA. Thus, the immune system harbors all of the necessary constituents for GABA signaling, and GABA itself may function as a paracrine or autocrine factor. These observations led us to ask further whether manipulation of the GABA pathway influences an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Increasing GABAergic activity ameliorates ongoing paralysis in EAE via inhibition of inflammation. GABAergic agents act directly on APCs, decreasing MAPK signals and diminishing subsequent adaptive inflammatory responses to myelin proteins.

Abstract

N-methyl-D-aspartate receptors (NMDARs) play important functions in neural development. NR2B is the predominant NR2 subunit of NMDAR in the developing brain. Here we use mosaic analysis with double markers (MADM) to knock out NR2B in isolated single cells and analyze its cell-autonomous function in dendrite development. NR2B mutant dentate gyrus granule cells (dGCs) and barrel cortex layer 4 spiny stellate cells (bSCs) have similar dendritic growth rates, total length, and branch number as control cells. However, mutant dGCs maintain supernumerary primary dendrites resulting from a pruning defect. Furthermore, while control bSCs restrict dendritic growth to a single barrel, mutant bSCs maintain dendritic growth in multiple barrels. Thus, NR2B functions cell autonomously to regulate dendrite patterning to ensure that sensory information is properly represented in the cortex. Our study also indicates that molecular mechanisms that regulate activity-dependent dendrite patterning can be separated from those that control general dendrite growth and branching.

Abstract

Vesicular secretion of neurotransmitter is essential for neuronal communication. Kiss-and-run is a mode of membrane fusion and retrieval without the full collapse of the vesicle into the plasma membrane and de novo regeneration. The importance of kiss-and-run during efficient neurotransmission has remained in doubt. We developed an approach for loading individual synaptic vesicles with single quantum dots. Their size and pH-dependent photoluminescence change allowed us to distinguish kiss-and-run from full-collapse fusion and to track single vesicles through multiple rounds of kiss-and-run and reuse, without perturbing vesicle cycling. Kiss-and-run dominated at the beginning of stimulus trains, reflecting the preference of vesicles with high release probability. Its incidence was increased by rapid firing, a response appropriate to shape the kinetics of neurotransmission during a wide range of firing patterns.

Abstract

Communication between cell surface proteins and the nucleus is integral to many cellular adaptations. In the case of ion channels in excitable cells, the dynamics of signaling to the nucleus are particularly important because the natural stimulus, surface membrane depolarization, is rapidly pulsatile. To better understand excitation-transcription coupling we characterized the dependence of cAMP response element-binding protein phosphorylation, a critical step in neuronal plasticity, on the level and duration of membrane depolarization. We find that signaling strength is steeply dependent on depolarization, with sensitivity far greater than hitherto recognized. In contrast, graded blockade of the Ca(2+) channel pore has a remarkably mild effect, although some Ca(2+) entry is absolutely required. Our data indicate that Ca(2+)/CaM-dependent protein kinase II acting near the channel couples local Ca(2+) rises to signal transduction, encoding the frequency of Ca(2+) channel openings rather than integrated Ca(2+) flux-a form of digital logic.

Abstract

Synaptic homeostasis, induced by chronic changes in neuronal activity, is well studied in cultured neurons, but not in more physiological networks where distinct synaptic circuits are preserved. We characterized inactivity-induced adaptations at three sets of excitatory synapses in tetrodotoxin-treated organotypic hippocampal cultures. The adaptation to inactivity was strikingly synapse specific. Hippocampal throughput synapses (dentate-to-CA3 and CA3-to-CA1) were upregulated, conforming to homeostatic gain control in order to avoid extreme limits of neuronal firing. However, chronic inactivity decreased mEPSC frequency at CA3-to-CA3 synapses, which were isolated pharmacologically or surgically. This downregulation of recurrent synapses was opposite to that expected for conventional homeostasis, in apparent conflict with typical gain control. However, such changes contributed to an inactivity-induced shortening of reverberatory bursts generated by feedback excitation among CA3 pyramids, safeguarding the network from possible runaway excitation. Thus, synapse-specific adaptations of synaptic weight not only contributed to homeostatic gain control, but also dampened epileptogenic network activity.

The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of Ca(V)1.2 L-type calcium channelsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABarrett, C. F., Tsien, R. W.2008; 105 (6): 2157-2162

Abstract

Calcium entry into excitable cells is an important physiological signal, supported by and highly sensitive to the activity of voltage-gated Ca2+ channels. After membrane depolarization, Ca2+ channels first open but then undergo various forms of negative feedback regulation including voltage- and calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca2+ channel activity is perturbed in a rare yet devastating disorder known as Timothy syndrome (TS), whose features include autism or autism spectrum disorder along with severe cardiac arrhythmia and developmental abnormalities. Most cases of TS arise from a sporadic single nucleotide change that generates a mutation (G406R) in the pore-forming subunit of the L-type Ca2+ channel Ca(V)1.2. We found that the TS mutation powerfully and selectively slows VDI while sparing or possibly speeding the kinetics of CDI. The deceleration of VDI was observed when the L-type channels were expressed with beta1 subunits prominent in brain, as well as beta2 subunits of importance for the heart. Dissociation of VDI and CDI was further substantiated by measurements of Ca2+ channel gating currents and by analysis of another channel mutation (I1624A) that hastens VDI, acting upstream of the step involving Gly406. As highlighted by the TS mutation, CDI does not proceed to completeness but levels off at approximately 50%, consistent with a change in gating modes and not an absorbing inactivation process. Thus, the TS mutation offers a unique perspective on mechanisms of inactivation as well as a promising starting point for exploring the underlying pathophysiology of autism.

Quantum dots provide an optical signal specific to full collapse fusion of synaptic vesiclesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAZhang, Q., Cao, Y., Tsien, R. W.2007; 104 (45): 17843-17848

Abstract

Synaptic vesicles are responsible for releasing neurotransmitters and are thus essential to brain function. The classical mode of vesicle recycling includes full collapse of the vesicle into the plasma membrane and clathrin-mediated regeneration of a new vesicle. In contrast, a nonclassical mode known as "kiss-and-run" features fusion by a transient fusion pore without complete loss of vesicle identity and offers possible advantages for increasing the throughput of neurotransmission. Studies of vesicular traffic have benefited greatly from fluorescent probes like FM dyes and synaptopHluorin. However, intrinsic properties of these probes limit their ability to provide a simple and precise distinction between classical and nonclassical modes. Here we report a novel optical probe specific to full collapse fusion, capitalizing on the size and superior photo-properties of photoluminescent quantum dots (Qdots). Qdots with exposed carboxyl groups were readily taken up by synaptic vesicles in an activity-, Ca(2+)-, and clathrin-dependent manner. Electron microscopy showed that Qdots were harbored within individual vesicles in a 1:1 ratio. The release of Qdots was activity- and Ca(2+)-dependent, similar to FM dyes. As artificial cargo, approximately 15 nm in diameter, Qdots will not escape vesicles during kiss-and-run but only with full collapse fusion. Strikingly, Qdots unloaded with kinetics substantially slower than destaining of FM dye, indicating that full-collapse fusion contributed only a fraction of all fusion events. As a full-collapse-fusion-responsive reporter, Qdots will likely promote better understanding of vesicle recycling at small CNS nerve terminals.

Abstract

The sympathetic nervous system regulates cardiac function through the activation of adrenergic receptors (ARs). beta(1) and beta(2)ARs are the primary sympathetic receptors in the heart and play different roles in regulating cardiac contractile function and remodeling in response to injury. In this study, we examine the targeting and trafficking of beta(1) and beta(2)ARs at cardiac sympathetic synapses in vitro. Sympathetic neurons form functional synapses with neonatal cardiac myocytes in culture. The myocyte membrane develops into specialized zones that surround contacting axons and contain accumulations of the scaffold proteins SAP97 and AKAP79/150 but are deficient in caveolin-3. The beta(1)ARs are enriched within these zones, whereas beta(2)ARs are excluded from them after stimulation of neuronal activity. The results indicate that specialized signaling domains are organized in cardiac myocytes at sites of contact with sympathetic neurons and that these domains are likely to play a role in the subtype-specific regulation of cardiac function by beta(1) and beta(2)ARs in vivo.

Abstract

LTP and other rapidly induced forms of synaptic modification tune individual synaptic weights, whereas slower forms of plasticity such as adaptation to inactivity are thought to keep neurons within their firing limits and preserve their capability for information processing. Here we describe progress in understanding the relationship between LTP and adaptation to inactivity. A prevailing view is that adaptation to inactivity is purely postsynaptic, scales synaptic strength uniformly across all synapses, and thus preserves relative synaptic weights without interfering with signatures of prior LTP or the relative capacity for future LTP. However, recent evidence in hippocampal neurons indicates that, like LTP, adaptation to AMPA receptor blockade can draw upon a repertoire of synaptic expression mechanisms including enhancement of presynaptic vesicular turnover and increased quantal amplitude mediated by recruitment of homomeric GluR1 AMPA receptors. These pre- and postsynaptic changes appeared coordinated and preferentially expressed at subset of synapses, thereby increasing the variability of miniature EPSCs. In contrast to the NMDA receptor-, Ca2+ entry-dependent induction of LTP, adaptation to inactivity may be mediated by attenuation of voltage-sensitive L-type Ca2+ channel function. The associated intracellular signaling involves elevation of betaCaMKII, which in turn downregulates alphaCaMKII, a key player in LTP. Thus, adaptation to inactivity and LTP are not strictly independent with regard to mechanisms of signaling and expression. Indeed, we and others have found that responses to LTP-inducing stimuli can be sharply altered by prior inactivity, suggesting that the slow adaptation changes the rules of plasticity-an interesting example of "metaplasticity".

Abstract

The number and diversity of plasticity mechanisms in the brain raises a central question: does a neural circuit store all memories by stereotyped application of the available plasticity mechanisms, or can subsets of these mechanisms be selectively engaged for specific memories? The uniform architecture of the cerebellum has inspired the idea that plasticity mechanisms like cerebellar long-term depression (LTD) contribute universally to memory storage. To test this idea, we investigated a set of closely related, cerebellum-dependent motor memories. In mutant mice lacking Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), the maintenance of cerebellar LTD is abolished. Although memory for an increase in the gain of the vestibulo-ocular reflex (VOR) induced with high-frequency stimuli was impaired in these mice, memories for decreases in VOR gain and increases in gain induced with low-frequency stimuli were intact. Thus, a particular plasticity mechanism need not support all cerebellum-dependent memories, but can be engaged selectively according to the parameters of training.

Abstract

Nicotinic acetylcholine receptors (nAChRs) are inhibited by several drugs that are commonly thought to be specific for L-type calcium channels (LTCCs). In neurons, LTCCs are activated by nicotine-induced depolarization to engage downstream signaling events; however, the role of LTCC drug interactions with nAChRs in signaling has not been examined in detail. We investigated the effects of LTCC ligands on nAChR currents and downstream signaling in rat superior cervical ganglion (SCG) neurons. We found that 10microM nicotine and 40mM K(+) both reversibly depolarize SCG neurons to -20mV, sufficient to activate LTCCs and downstream signaling, including induction of nuclear phospho-CREB (pCREB); this induction was blocked by LTCC antagonists. Interestingly, the effects of LTCC antagonists on nicotine-induced signaling to CREB are not mediated by their actions on LTCCs, but rather via inhibition of nAChRs, which prevents nicotine-induced depolarization. We show that this effect is sufficient to block pCREB induction in neurons expressing an antagonist-insensitive LTCC. Taken together, our data show that, at concentrations typically used to block LTCCs, these antagonists inhibit nAChR currents and downstream signaling. These findings serve as a caution in attributing a role for LTCCs when using these drugs experimentally or therapeutically.

Abstract

Neurotransmitters and hormones are released from neurosecretory cells by exocytosis (fusion) of synaptic vesicles, large dense-core vesicles and other types of vesicles or granules. The exocytosis is terminated and followed by endocytosis (retrieval). More than fifty years of research have established full-collapse fusion and clathrin-mediated endocytosis as essential modes of exo-endocytosis. Kiss-and-run and vesicle reuse represent alternative modes, but their prevalence and importance have yet to be elucidated, especially in neurons of the mammalian CNS. Here we examine various modes of exo-endocytosis across a wide range of neurosecretory systems. Full-collapse fusion and kiss-and-run coexist in many systems and play active roles in exocytotic events. In small nerve terminals of CNS, kiss-and-run has an additional role of enabling nerve terminals to conserve scarce vesicular resources and respond to high-frequency inputs. Full-collapse fusion and kiss-and-run will each contribute to maintaining cellular communication over a wide range of frequencies.

Abstract

The kinetics of exo-endocytotic recycling could restrict information transfer at central synapses if neurotransmission were entirely reliant on classical full-collapse fusion. Nonclassical fusion retrieval by kiss-and-run would be kinetically advantageous but remains controversial. We used a hydrophilic quencher, bromophenol blue (BPB), to help detect nonclassical events. Upon stimulation, extracellular BPB entered synaptic vesicles and quenched FM1-43 fluorescence, indicating retention of FM dye beyond first fusion. BPB also quenched fluorescence of VAMP (synaptobrevin-2)-EGFP, thus indicating the timing of first fusion of vesicles in the total recycling pool. Comparison with FM dye destaining revealed that kiss-and-run strongly prevailed over full-collapse fusion at low frequency, giving way to a near-even balance at high frequency. Quickening of kiss-and-run vesicle reuse was also observed at higher frequency in the average single vesicle fluorescence response. Kiss-and-run and reuse could enable hippocampal nerve terminals to conserve scarce vesicular resources when responding to widely varying input patterns.

Abstract

Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Ca(v)1.2) contributes to the positive force-frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit alpha(1C) (Ca(v)alpha1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of alpha1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to alpha1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.

Abstract

In response to activity deprivation, CNS neurons undergo slow adaptive modification of unitary synaptic transmission. The changes are comparable in degree to those induced by brief intense stimulation, but their molecular basis is largely unknown. Our data indicate that prolonged AMPAR blockade acts through loss of Ca2+ entry through L-type Ca2+ channels to bring about an increase in both vesicle pool size and turnover rate, as well as a postsynaptic enhancement of the contribution of GluR1 homomers, concentrated at the largest synapses. The changes were consistent with a morphological scaling of overall synapse size, but also featured a dramatic shift toward synaptic drive contributed by the Ca2+-permeable homomeric GluR1 receptors. These results extend beyond "synaptic homeostasis" to involve more profound changes that can be better described as "metaplasticity".

Abstract

Familial hemiplegic migraine type 1 (FHM1) arises from missense mutations in the gene encoding alpha1A, the pore-forming subunit of P/Q-type calcium channels. The nature of the channel disorder is fundamental to the disease, yet is not well understood. We studied how the most prevalent FHM1 mutation, a threonine to methionine substitution at position 666 (TM), affects both ionic current and gating current associated with channel activation, a previously unexplored feature of P/Q channels. Whole-cell currents were measured in HEK293 cells expressing channels containing either wild-type (WT) or TM alpha1A. Calcium currents were significantly smaller in cells expressing TM channels, consistent with previous reports. In contrast, surface expression of TM channels, measured by immunostaining against an extracellular epitope, was not decreased, and Western blots demonstrated that TM alpha1A subunits were expressed as full-length proteins. WT and TM gating currents were isolated by replacing Ca2+ with the nonpermeant cation La3+. The gating currents generated by the mutant channels were one-third that of WT, a deficiency sufficient to account for the observed attenuation in calcium current; the remaining gating current was no different in kinetics or voltage dependence. Thus, the decreased calcium influx seen with TM channels can be attributed to a reduced number of channels available to undergo the voltage-dependent conformational changes needed for channel opening, not to fewer channel proteins expressed on the cell surface. This identification of an intrinsic defect in FHM1 mutant channels helps explain their impact on neurotransmission when they occupy type-specific slots for P/Q channels at central nerve terminals.

Effects of familial hemiplegic migraine type 1 mutations on neuronal P/Q-type Ca2+ channel activity and inhibitory synaptic transmissionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACao, Y. Q., Tsien, R. W.2005; 102 (7): 2590-2595

Abstract

Inhibitory synapses play key roles in the modulatory circuitry that regulates pain signaling and generation of migraine headache. A rare, dominant form of this common disease, familial hemiplegic migraine type 1 (FHM1), arises from missense mutations in the pore-forming alpha1A subunit of P/Q-type Ca2+ channels. These channels are normally vital for presynaptic Ca2+ entry and neurotransmitter release at many central synapses, raising questions about effects of FHM1 mutations on neuronal Ca2+ influx and inhibitory and excitatory neurotransmission. We have expressed the four original FHM1 mutant channels in hippocampal neurons from alpha1A knockout mice. Whole-cell recordings indicated that FHM1 mutant channels were less effective than wild-type channels in their ability to conduct P/Q-type current, but not generally different from wild type in voltage-dependent channel gating. Ca2+ influx triggered by action potential waveforms was also diminished. In keeping with decreased channel activity, FHM1 mutant channels were correspondingly impaired in supporting the P/Q-type component of inhibitory neurotransmission. When expressed in wild-type inhibitory neurons, FHM1 mutant channels reduced the contribution of P/Q-type channels to GABAergic synaptic currents, consistent with a competition of mutant and endogenous channels for P/Q-specific slots. In all cases, N-type channels took up the burden of supporting transmission and homeostatic mechanisms maintained overall synaptic strength. The shift to reliance on N-type channels greatly increased the susceptibility to G protein-coupled modulation of neurotransmission, studied with the GABAB agonist baclofen. Thus, mutant-expressing synapses might be weakened in a heightened state of neuromodulation like that provoked by triggers of migraine such as stress.

Abstract

Several human channelopathies result from mutations in alpha1A, the pore-forming subunit of P/Q-type Ca2+ channels, conduits of presynaptic Ca2+ entry for evoked neurotransmission. We found that wild-type human alpha1A subunits supported transmission between cultured mouse hippocampal neurons equally well as endogenous mouse alpha1A, whereas introduction of impermeant human alpha1A hampered the effect of endogenous subunits. Thus, presynaptic P/Q-type channels may compete for channel type-preferring "slots" that limit their synaptic effectiveness. The existence of slots generates predictions for how neurotransmission might be affected by changes in Ca2+ channel properties, which we tested by studying alpha1A mutations that are associated with familial hemiplegic migraine type 1 (FHM1). Mutant human P/Q-type channels were impaired in contributing to neurotransmission in precise accord with their deficiency in supporting whole-cell Ca2+ channel activity. Expression of mutant channels in wild-type neurons reduced the synaptic contribution of P/Q-type channels, suggesting that competition for type-preferring slots might support the dominant inheritance of FHM1.

Abstract

At central synapses, P/Q-type Ca(2+) channels normally provide a critical Ca(2+) entry pathway for neurotransmission. Nevertheless, we found that nerve terminals lacking alpha(1A) (Ca(V)2.1), the pore-forming subunit of P/Q-type channels, displayed a remarkable preservation of synaptic function. Two consistent physiological changes reflective of synaptic homeostasis were observed in cultured hippocampal neurons derived from alpha(1A) (-/-) mice. First, the presynaptic response to an ionophore-mediated Ca(2+) elevation was 50% greater, indicating an enhanced Ca(2+) sensitivity of the release machinery. Second, basal miniature excitatory postsynaptic current frequency in alpha(1A) (-/-) neurons was increased 2-fold compared with WT neurons and occluded the normal response of presynaptic terminals to cAMP elevation, suggesting that the compensatory mechanism in alpha(1A) (-/-) synapses and the modulation of presynaptic function by PKA might share a final common pathway. We used cDNA microarray analysis to identify molecular changes underlying homeostatic regulation in the alpha(1A) (-/-) hippocampus. The 40,000 entries in our custom-made array included likely targets of presynaptic homeostasis, along with many other transcripts, allowing a wide-ranging examination of gene expression. The developmental pattern of changes in transcript levels relative to WT was striking; mRNAs at 5 and 11 days postnatal showed little deviation, but clear differences emerged by 22 days. Many of the transcripts that differed significantly in abundance corresponded to known genes that could be incorporated within a logical pattern consistent with the modulation of presynaptic function. Changes in endocytotic proteins, signal transduction kinases, and candidates for Ca(2+)-sensing molecules were consistent with implications of the direct physiological experiments.

Paired-pulse depression of unitary quantal amplitude at single hippocampal synapsesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChen, G., Harata, N. C., Tsien, R. W.2004; 101 (4): 1063-1068

Abstract

At central synapses, quantal size is generally regarded as fluctuating around a fixed mean with little change during short-term synaptic plasticity. We evoked quantal release by brief electric stimulation at single synapses visualized with FM 1-43 dye in hippocampal cultures. The majority of quantal events evoked at single synapses were monovesicular, based on examination of amplitude distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor-mediated responses. Consistent with previous findings, the quantal size did not change during paired-pulse facilitation (PPF), supporting the notion that the evoked events were monoquantal. However, during paired-pulse depression (PPD), there was a significant decrease in unitary quantal size, which was not due to postsynaptic receptor desensitization. This asymmetry of quantal modulation during PPF and PPD was demonstrated at the same single synapse at different extracellular calcium concentrations. Our results indicate that PPF can be fully accounted for by an increase of release probability, whereas PPD may be caused by decreases in both release probability and quantal size. One possible explanation is that the release of a quantum of neurotransmitter from synaptic vesicles is not invariant but subject to rapid calcium-dependent modulation during short-term synaptic plasticity.

Abstract

Synaptic activity causes reductions in cleft [Ca(2+)] that may impact subsequent synaptic efficacy. Using modified patch-clamp techniques to record from single neocortical nerve terminals, we report that physiologically relevant reductions of extracellular [Ca(2+)] ([Ca(2+)](o)) activate voltage-dependent outward currents. These outward currents are carried by a novel nonselective cation (NSC) channel that is indirectly inhibited by various extracellular agents (rank order potency, Gd(3+) > spermidine > Ca(2+) > Mg(2+), typical for [Ca(2+)](o) receptors). The identification of a Ca(2+) sensor-NSC channel pathway establishes the existence of a mechanism by which presynaptic terminals can detect and respond to reductions in cleft [Ca(2+)]. Activation of NSC channels by falls in [Ca(2+)](o) would be expected during periods of high activity in the neocortex and may modulate the excitability of the presynaptic terminal.

Abstract

At synapses of the mammalian central nervous system, release of neurotransmitter occurs at rates transiently as high as 100 Hz, putting extreme demands on nerve terminals with only tens of functional vesicles at their disposal. Thus, the presynaptic vesicle cycle is particularly critical to maintain neurotransmission. To understand vesicle cycling at the most fundamental level, we studied single vesicles undergoing exo/endocytosis and tracked the fate of newly retrieved vesicles. This was accomplished by minimally stimulating boutons in the presence of the membrane-fluorescent styryl dye FM1-43, then selecting for terminals that contained only one dye-filled vesicle. We then observed the kinetics of dye release during single action potential stimulation. We found that most vesicles lost only a portion of their total dye during a single fusion event, but were able to fuse again soon thereafter. We interpret this as direct evidence of "kiss-and-run" followed by rapid reuse. Other interpretations such as "partial loading" and "endosomal splitting" were largely excluded on the basis of multiple lines of evidence. Our data placed an upper bound of <1.4 s on the lifetime of the kiss-and-run fusion event, based on the assumption that aqueous departitioning is rate limiting. The repeated use of individual vesicles held over a range of stimulus frequencies up to 30 Hz and was associated with neurotransmitter release. A small percentage of fusion events did release a whole vesicle's worth of dye in one action potential, consistent with a classical picture of exocytosis as fusion followed by complete collapse or at least very slow retrieval.

Abstract

Vesicle fusion and recycling are particularly critical for ongoing neurotransmitter release in the small nerve terminals of the brain, which typically contain about 30 functional vesicles. However, the modes of exocytosis and endocytosis that operate at synapses of the central nervous system are incompletely understood. Here we show real-time visualization of a single vesicle fusing at a small synapse of the central nervous system, made possible by highly intensified charge-coupled device imaging of hippocampal synaptic terminals, in which a single vesicle was labelled with the fluorescent membrane marker FM1-43 (ref. 6). In a small number of cases, full loss of fluorescent membrane dye was elicited by a single action potential, consistent with classical complete collapse. In most cases, however, action potentials triggered only partial loss of fluorescence, suggesting vesicular retention of membrane marker, consistent with 'kiss-and-run' vesicle cycling. An alternative hypothesis of independent fusion of partially stained vesicles arising from endosomal splitting could be excluded by observations on the size and timing of successive fusion events. Thus, our experimental evidence supports a predominance of kiss-and-run fusion events and rapid vesicular re-use.

Abstract

Signaling from synapse to nucleus is vital for activity-dependent control of neuronal gene expression and represents a sophisticated form of neural computation. The nature of specific signal initiators, nuclear translocators and effectors has become increasingly clear, and supports the idea that the nucleus is able to make sense of a surprising amount of fast synaptic information through intricate biochemical mechanisms. Information transfer to the nucleus can be conveyed by physical translocation of messengers at various stages within the multiple signal transduction cascades that are set in motion by a Ca(2+) rise near the surface membrane. The key role of synapse-to-nucleus signaling in circadian rhythms, long-term memory, and neuronal survival sheds light on the logical underpinning of these signaling mechanisms.

Abstract

Transmission at the mouse neuromuscular junction normally relies on P/Q-type channels, but became jointly dependent on both N- and R-type Ca(2+) channels when the PQ-type channel alpha(1A) subunit was deleted. R-type channels lay close to Ca(2+) sensors for exocytosis and I(K(Ca)) channel activation, like the P/Q-type channels they replaced. In contrast, N-type channels were less well localized, but abundant enough to influence secretion strongly, particularly when action potentials were prolonged. Our data suggested that active zone structures may select among multiple Ca(2+) channels in the hierarchy P/Q >R >N. The alpha(1A)-/- neuromuscular junction displayed several other differences from wild-type: lowered quantal content but greater ability to withstand reductions in the Ca(2+)/Mg(2+) ratio, and little or no paired-pulse facilitation, the latter findings possibly reflecting compensatory mechanisms at individual release sites. Changes in presynaptic function were also associated with a significant reduction in the size of postsynaptic acetylcholine receptor clusters.

Abstract

We show that alpha and betaCaMKII are inversely regulated by activity in hippocampal neurons in culture: the alpha/beta ratio shifts toward alpha during increased activity and beta during decreased activity. The swing in ratio is approximately 5-fold and may help tune the CaMKII holoenzyme to changing intensities of Ca(2+) signaling. The regulation of CaMKII levels uses distinguishable pathways, one responsive to NMDA receptor blockade that controls alphaCaMKII alone, the other responsive to AMPA receptor blockade and involving betaCaMKII and possibly further downstream effects of betaCaMKII on alphaCaMKII. Overexpression of alphaCaMKII or betaCaMKII resulted in opposing effects on unitary synaptic strength as well as mEPSC frequency that could account in part for activity-dependent effects observed with chronic blockade of AMPA receptors. Regulation of CaMKII subunit composition may be important for both activity-dependent synaptic homeostasis and plasticity.

Abstract

Pore size is of considerable interest in voltage-gated Ca(2+) channels because they exemplify a fundamental ability of certain ion channels: to display large pore diameter, but also great selectivity for their ion of choice. We determined the pore size of several voltage-dependent Ca(2+) channels of known molecular composition with large organic cations as probes. T-type channels supported by the Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 subunits; L-type channels encoded by the Ca(V)1.2, beta(1), and alpha(2)delta(1) subunits; and R-type channels encoded by the Ca(V)2.3 and beta(3) subunits were each studied using a Xenopus oocyte expression system. The weak permeabilities to organic cations were resolved by looking at inward tails generated upon repolarization after a large depolarizing pulse. Large inward NH(4)(+) currents and sizable methylammonium and dimethylammonium currents were observed in all of the channels tested, whereas trimethylammonium permeated only through L- and R-type channels, and tetramethylammonium currents were observed only in L-type channels. Thus, our experiments revealed an unexpected heterogeneity in pore size among different Ca(2+) channels, with L-type channels having the largest pore (effective diameter = 6.2 A), T-type channels having the tiniest pore (effective diameter = 5.1 A), and R-type channels having a pore size intermediate between these extremes. These findings ran counter to first-order expectations for these channels based simply on their degree of selectivity among inorganic cations or on the bulkiness of their acidic side chains at the locus of selectivity.

Abstract

Synapse-to-nucleus signaling leading to CREB-mediated transcription is important for neuronal plasticity. Nuclear CREB phosphorylation at Ser133 allows convergence of multiple kinase pathways driven by neuronal activity and links them to transcriptional activation. But, can various pathways share a common effector mechanism (phosphorylating Ser133) while generating distinct patterns of gene expression? We review three Neuron articles that highlight novel ways Ca(2+) signals can trigger multiple phosphorylation events working in combination to control CREB and its interaction with coactivator molecules.

Calmodulin priming: Nuclear translocation of a calmodulin complex and the memory of prior neuronal activityPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMermelstein, P. G., Deisseroth, K., Dasgupta, N., Isaksen, A. L., Tsien, R. W.2001; 98 (26): 15342-15347

Abstract

The neuronal nucleus plays a vital role in information processing, but whether it supports computational functions such as paired-pulse facilitation, comparable to synapses, is unclear. Ca(2+)-dependent movement of calmodulin (CaM) to the nucleus is highly responsive to Ca(2+) entry through L-type channels and promotes activation of the transcription factor CREB (cAMP-responsive element binding protein) through phosphorylation by CaM-sensitive kinases. We characterized key features of this CaM translocation and its possible role in facilitation of nuclear signaling. Nuclear CaM was elevated within 15 s of stimulus onset, preceding the first signs of CREB phosphorylation in hippocampal pyramidal neurons. Depolarization-induced elevation of nuclear CaM also was observed in cerebellar granule cells, neocortical neurons, and dentate gyrus granule cells. Nuclear translocation of CaM was not blocked by disruption of actin filaments or microtubules, or by emptying endoplasmic reticulum Ca(2+) stores with thapsigargin. Translocation of fluorescently tagged CaM was prevented by fusing it with the Ca(2+)/CaM binding peptide M13, suggesting that nuclear CaM accumulation depends on association with endogenous Ca(2+)/CaM binding proteins. To determine whether increased nuclear [CaM] might influence subsequent nuclear signal processing, we compared responses to two consecutive depolarizing stimuli. After a weak "priming" stimulus that caused CaM translocation, CREB phosphorylation caused by a subsequent stimulus was significantly faster, more sensitive to Ca(2+) elevation, and less specifically dependent on Ca(2+) influx through L-type channels. CaM translocation not only supports rapid signaling to the nucleus, but also could provide a "memory" for facilitatory effects of repeated neural activity, seen in altered phosphorylated CREB dynamics and Ca(2+) channel dependence.

Abstract

The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo-endocytotic recycling following stimulated Ca(2+) entry. This recycling pool typically contained 30-45 vesicles, only a minority fraction (15-20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.

Abstract

Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3'-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC-). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.

Abstract

Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.

Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathwayPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAWu, G. Y., Deisseroth, K., Tsien, R. W.2001; 98 (5): 2808-2813

Abstract

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca(2+) transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca(2+) signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only approximately 1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Abstract

Memory storage in mammalian neurons probably depends on both biochemical events and morphological alterations in dendrites. Here we report an activity-dependent stabilization of the MAP kinase (MAPK) pathway, prominent in hippocampal dendrites. The longevity of the signal in these dendrites was increased to hours when multiple spaced stimuli were used. Likewise, spaced stimuli and MAPK activation were critical for protrusion of new dendritic filopodia that also remained stable for hours. Our experiments define a new role for stimulus-specific responses of MAPK signaling in activity-dependent neuronal plasticity. The local biochemical signaling in dendrites complements MAPK signaling in gene expression. Together, these processes may support long-lasting behavioral changes.

Abstract

Functional presynaptic vesicles have been subdivided into readily releasable (RRP) and reserve (RP) pools. We studied recycling properties of RRP vesicles through differential retention of FM1-43 and FM2-10 and by varying the time window for FM dye uptake. Both approaches indicated that vesicles residing in the RRP underwent rapid endocytosis (tau approximately 1s), whereas newly recruited RP vesicles were recycled slowly (tau approximately 30 s). With repeated challenges (hypertonic or electrical stimuli), the ability to release neurotransmitter recovered 10-fold more rapidly than restoration of FM2-10 destaining. Finding neurotransmission in the absence of destaining implied that rapidly endocytosed RRP vesicles were capable of reuse, a process distinct from repopulation from the RP. Reuse would greatly expand the functional capabilities of a limited number of vesicles in CNS terminals, particularly during intermittent bursts of activity.

Abstract

Syntaxin, a membrane protein vital in triggering vesicle fusion, interacts with voltage-gated N- and P/Q-type Ca(2+) channels. This biochemical association is proposed to colocalize Ca(2+) channels and presynaptic release sites, thus supporting rapid and efficient initiation of neurotransmitter release. The syntaxin channel interaction may also support a novel signaling function, to modulate Ca(2+) channels according to the state of the associated release machinery (Bezprozvanny et al., 1995; Wiser et al., 1996; see also Mastrogiacomo et al., 1994). Here we report that syntaxin 1A (syn1A) coexpressed with N-type channels in Xenopus oocytes greatly promoted slow inactivation gating, but had little or no effect on the onset of and recovery from fast inactivation. Accordingly, the effectiveness of syntaxin depended strongly on voltage protocol. Slow inactivation was found for N-type channels even in the absence of syntaxin and could be distinguished from fast inactivation on the basis of its slow kinetics, distinct voltage dependence (voltage-independent at potentials higher than the level of half-inactivation), and temperature independence (Q(10), approximately 0.8). Trains of action potential-like stimuli were more effective than steady depolarizations in stabilizing the slowly inactivated condition. Agents that stimulate protein kinase C decreased the inhibitory effect of syntaxin on N-type channels. Application of BoNtC1 to cleave syntaxin sharply attenuated the modulatory effects on Ca(2+) channel gating, consistent with structural analysis of syntaxin modulation, supporting use of this toxin to test for the impact of syntaxin on Ca(2+) influx in nerve terminals.

Abstract

When the presynaptic membrane protein syntaxin is coexpressed in Xenopus oocytes with N- or P/Q-type Ca(2+) channels, it promotes their inactivation (Bezprozvanny et al., 1995; Wiser et al., 1996, 1999; Degtiar et al., 2000) (I. B. Bezprozvanny, P. Zhong, R. H. Scheller, and R. W. Tsien, unpublished observations). These findings led to the hypothesis that syntaxin influences Ca(2+) channel function in presynaptic endings, in a reversal of the conventional flow of information from Ca(2+) channels to the release machinery. We examined this effect in isolated mammalian nerve terminals (synaptosomes). Botulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied to rat neocortical synaptosomes at concentrations that completely blocked neurotransmitter release. This treatment altered the pattern of Ca(2+) entry monitored with fura-2. Whereas the initial Ca(2+) rise induced by depolarization with K(+)-rich solution was unchanged, late Ca(2+) entry was strongly augmented by syntaxin cleavage. Similar results were obtained when Ca(2+) influx arose from repetitive firing induced by the K(+)-channel blocker 4-aminopyridine. Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 with BoNtE failed to produce a significant change in Ca(2+) entry. The BoNtC1-induced alteration in Ca(2+) signaling was specific to voltage-gated Ca(2+) channels, not Ca(2+) extrusion or buffering, and it involved N-, P/Q- and R-type channels, the high voltage-activated channels most intimately associated with presynaptic release machinery. The modulatory effect of syntaxin was not immediately manifest when synaptosomes had been K(+)-predepolarized in the absence of external Ca(2+), but developed with a delay after admission of Ca(2+), suggesting that vesicular turnover may be necessary to make syntaxin available for its stabilizing effect on Ca(2+) channel inactivation.

Abstract

Activity-dependent gene expression in neurons shows a remarkable ability to differentiate between different types of stimulation: orthodromic inputs that engage synaptic transmission are much more effective than antidromic stimuli that do not. We have studied the basis of such selectivity in cultured hippocampal neurons in which nuclear cAMP response element-binding protein (CREB) phosphorylation is induced by synaptic activity but not by action potential (AP) stimulation in the absence of EPSPs, although spikes by themselves generate large elevations in intracellular Ca(2+). Previous work has shown that Ca(2+) entry through L-type Ca(2+) channels plays a dominant role in triggering calmodulin mobilization and activation of calmodulin-dependent kinases that phosphorylate CREB, raising the possibility that L-type channels contribute to the selective response to EPSPs rather than APs. Accordingly, we performed voltage-clamp experiments to compare the currents carried by L-type channels during depolarizing waveforms that approximated APs or dendritic EPSPs. The integrated current generated by L-type channels was significantly less after mock APs than with EPSP-like depolarizations. The difference was traced to two distinct factors. Compared with other channels, L-type channels activated at relatively negative potentials, favoring their opening with EPSP stimulation; they also exhibited relatively slow activation kinetics, weighing against their contribution during an AP. The relative ineffectiveness of APs as a stimulus for CREB phosphorylation could be overcome by exposure to the agonist Bay K8644, which potentiated the AP-induced influx through L-type channels by approximately 10-fold. Under normal conditions, the unique biophysical properties of L-type channels allow them to act as a kinetic filter to support spike-EPSP discrimination.

Abstract

Fluorescence imaging of presynaptic uptake and release of styryl dyes such as FM1-43 has provided valuable insights into synaptic function. However, in studies of CNS neurons, the utility of these dyes has been severely limited by nonsynaptic background fluorescence. This has thwarted the use of FM dyes in systems more intact than dissociated neuronal cultures. Here, we describe an approach to selectively reduce undesired fluorescence through quenching of the surface-bound FM1-43 signal. The introduction of sulforhodamine, a fluorophore that is not taken up by synaptic vesicles, selectively reduced the nonsynaptic fluorescence in FM1-43-labeled hippocampal cultures. When applied to rat hippocampal slices, this procedure allowed us to observe activity-dependent staining and destaining of functional synapses. Extending the usefulness of styryl dyes to slice preparations may help make functional synaptic networks amenable to optical measurements.

Activity-dependent regulation of synaptic clustering in a hippocampal culture systemPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKavalali, E. T., Klingauf, J., Tsien, R. W.1999; 96 (22): 12893-12900

Abstract

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1-43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-D-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.

Abstract

The molecular basis of learning and memory has been the object of several recent advances, which have focused attention on calcium-regulated pathways controlling transcription. One of the molecules implicated by pharmacological, biochemical and genetic approaches is the calcium/calmodulin-regulated phosphatase, calcineurin. In lymphocytes, calcineurin responds to specific calcium signals and regulates expression of several immediate early genes by controlling the nuclear import of the NF-ATc family of transcription factors. Here we show that NF-ATc4/NF-AT3 in hippocampal neurons can rapidly translocate from cytoplasm to nucleus and activate NF-AT-dependent transcription in response to electrical activity or potassium depolarization. The calcineurin-mediated translocation is critically dependent on calcium entry through L-type voltage-gated calcium channels. GSK-3 can phosphorylate NF-ATc4, promoting its export from the nucleus and antagonizing NF-ATc4-dependent transcription. Furthermore, we show that induction of the inositol 1,4,5-trisphosphate receptor type 1 is controlled by the calcium/calcineurin/NF-ATc pathway. This provides a new perspective on the function of calcineurin in the central nervous system and indicates that NF-AT-mediated gene expression may be involved in the induction of hippocampal synaptic plasticity and memory formation.

Abstract

Regulation of synaptic transmission is a widespread means for dynamic alterations in nervous system function. In several cases, this regulation targets vesicular recycling in presynaptic terminals and may result in substantial changes in efficiency of synaptic transmission. Traditionally, experimental accessibility of the synaptic vesicle cycle in central neuronal synapses has been largely limited to the exocytotic side, which can be monitored with electrophysiological responses to neurotransmitter release. Recently, physiological measurements on the endocytotic portion of the cycle have been made possible by the introduction of styryl dyes such as FM1-43 as fluorescent markers for recycling synaptic vesicles. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was greatly speeded by exposure to staurosporine or elevated extracellular calcium. The effective time-constant for retrieval can be < 2 seconds under appropriate conditions. Thus, hippocampal synapses capitalize on efficient mechanisms for endocytosis and their vesicular retrieval is subject to modulatory control.

Abstract

Many neurons of the central and peripheral nervous systems display multiple high voltage-activated (HVA) Ca2+ currents, often classified as L-, N-, P-, Q, and R-type. The heterogeneous properties of these channels have been attributed to diversity in their pore-forming alpha 1, subunits, in association with various beta subunits. However, there are large gaps in understanding how individual subunits contribute to Ca2+ channel diversity. Here we describe experiments to investigate the roles of alpha 1E and beta 3 subunits in mammalian neurons. The alpha 1E subunit is the leading candidate to account for the R-type channel, the least understood of the various types of high voltage-activated Ca2+ channels. Incubation with alpha 1E antisense oligonucleotide caused a 53% decrease in the peak R-type current density, while no significant changes in the current expression were seen in sense oligonucleotide-treated cells. The specificity of the alpha 1E antisense oligonucleotides was supported by the lack of change in the amplitude of P/Q current. These results upheld the hypothesis that members of the E class of alpha 1 subunits support the high voltage-activated R-type current in cerebellar granule cells. We studied the role of the Ca2+ channel beta 3 subunit using a gene targeting strategy. In sympathetic beta 3-/- neurons, the L-type current was significantly reduced relative to wild type (wt). In addition, N-type Ca2+ channels made up a smaller proportion of the total Ca2+ current than in wt due to a lower N-type current density in a group of neurons with small total currents. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence. The absence of the beta 3 subunit was associated with a shift in the more depolarized component of the activation along the voltage axis toward more negative potentials. The overall conclusion is that deletion of the beta 3 subunit affects at least three distinct types of HVA Ca2+ channel, but no single type of channel is solely dependent on beta 3.

Abstract

Presynaptic nerve terminals often contain as few as a hundred vesicles and so must recycle them soon after exocytosis to preserve synaptic transmission and presynaptic morphology during repetitive firing. The kinetics and mechanisms of vesicular endocytosis and repriming have therefore been studied. Vesicles in hippocampal nerve terminals can become available to release their contents within approximately 40 s of the previous round of exocytosis. Studies using the styryl dye FM1-43 have estimated the time constant for endocytosis as approximately 20-30 s at least half of the total recycling time, which is much slower than endocytosis in other secretory systems. It seems paradoxical that the neurosecretory terminals that could benefit the most from rapid endocytosis do not use such a mechanism. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was much faster after exposure to staurosporine or elevated extracellular calcium. Thus hippocampal synapses take advantage of efficient mechanisms for endocytosis, and their vesicular retrieval is subject to modulatory control.

Antisense oligonucleotides against alpha(1E) reduce R-type calcium currents in cerebellar granule cellsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAPiedras-Renteria, E. S., Tsien, R. W.1998; 95 (13): 7760-7765

Abstract

Many neurons of the central nervous system display multiple high voltage-activated Ca2+ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the alpha1E subunit, which yields Ca2+ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against alpha1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6-8 days in vitro. Incubation with alpha1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from -10 +/- 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to -4.8 +/- 0.8 pA/pF (n = 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (-11.3 +/- 3.2 pA/pF). The specificity of the alpha1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against alpha1A did not affect R-type current expression (-11.5 +/- 1.7 and -11.7 +/- 1.7 pA/pF, respectively), whereas the alpha1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of alpha1 subunits support the high voltage-activated R-type current in cerebellar granule cells.

Abstract

Activation of the transcription factor CREB is thought to be important in the formation of long-term memory in several animal species. The phosphorylation of a serine residue at position 133 of CREB is critical for activation of CREB. This phosphorylation is rapid when driven by brief synaptic activity in hippocampal neurons. It is initiated by a highly local, rise in calcium ion concentrations near the cell membrane, but culminates in the activation of a specific calmodulin-dependent kinase known as CaMK IV, which is constitutively present in the neuronal nucleus. It is unclear how the signal is conveyed from the synapse to the nucleus. We show here that brief bursts of activity cause a swift (approximately 1 min) translocation of calmodulin from the cytoplasm to the nucleus, and that this translocation is important for the rapid phosphorylation of CREB. Certain Ca2+ entry systems (L-type Ca2+ channels and NMDA receptors) are able to cause mobilization of calmodulin, whereas others (N- and P/Q-type Ca2+ channels) are not. This translocation of calmodulin provides a form of cellular communication that combines the specificity of local Ca2+ signalling with the ability to produce action at a distance.

Abstract

Hydrogen ions reduce ion flux through voltage-gated Ca2+ channels by binding to a single protonation site with an unusually high pKa. Recent evidence localizes the protonation site to the same locus that supports high affinity Ca2+ binding and selectivity, a set of four conserved glutamate residues near the external mouth of the pore. Remaining controversy concerns the question of whether the protonation site arises from a single glutamate, Glu-1086 (EIII), or a combination of Glu-1086 and Glu-334 (EI) working in concert. We tested these hypotheses with individual Glu --> Asp substitutions. The Glu --> Asp replacements in repeats I and III stood out in two ways. First, in both EID and EIIID, protonation was destabilized relative to wild type, whereas it was unchanged in EIID and stabilized in EIVD. The changes in affinity were entirely due to alterations in H+ off-rate. Second, the ratio of protonated conductance to deprotonated conductance was significantly closer to unity for EID and EIIID than for wild-type channels or other Asp mutants. Both results support the idea that EI and EIII act together to stabilize a single titratable H+ ion and behave nearly symmetrically in influencing pore conductance. Neutralization of EIII by alanine replacement clearly failed to abolish susceptibility to protonation, indicating that no single glutamate was absolutely required. Taken together, all the evidence supports a model in which multiple carboxylates work in concert to form a single high affinity protonation site.

Abstract

The selective block of N-type Ca2+ channels by omega-conotoxins has been a hallmark of these channels, critical in delineating their biological roles and molecular characteristics. Here we report that the omega-conotoxin-channel interaction depends strongly on channel gating. N-type channels (alpha1B, alpha2, and beta1) expressed in Xenopus oocytes were blocked with a variety of omega-conotoxins, including omega-CTx-GVIA, omega-CTx-MVIIA, and SNX-331, a derivative of omega-CTx-MVIIC. Changes in holding potential (HP) markedly altered the severity of toxin block and the kinetics of its onset and removal. Notably, strong hyperpolarization renders omega-conotoxin block completely reversible. These effects could be accounted for by a modulated receptor model, in which toxin dissociation from the inactivated state is approximately 60-fold slower than from the resting state. Because omega-conotoxins act exclusively outside cells, our results suggest that voltage-dependent inactivation of Ca2+ channels must be associated with an externally detectable conformational change.

Abstract

Dendritic arbors are critical for the information processing capability of central neurons, but quantitative analysis of their membrane properties has been hampered by their geometrical complexity. Here, we have focused on an important source of Ca2+ entry in dendrites, the voltage-gated Ca2+ channels, by applying the whole-cell voltage-clamp technique to isolated dendritic segments ("dendrosomes") from rat hippocampal neurons. We found that low voltage-activated T-type Ca2+ channels provide a significantly larger fraction of the Ca2+ influx in dendrites than their counterparts in cell bodies. Surprisingly, 60%-70% of the high voltage-activated Ca2+ current in dendrosomes was N and P/Q type, and these channels were susceptible to neurotransmitter inhibition, suggesting a novel physiological role for G protein-regulated Ca2+ channel modulation in controlling dendritic excitability and Ca2+ signaling.

Abstract

While changes in gene expression are critical for many brain functions, including long-term memory, little is known about the cellular processes that mediate stimulus-transcription coupling at central synapses. In studying the signaling pathways by which synaptic inputs control the phosphorylation state of cyclic AMP-responsive element binding protein (CREB) and determine expression of CRE-regulated genes, we found two important Ca2+/calmodulin (CaM)-regulated mechanisms in hippocampal neurons: a CaM kinase cascade involving nuclear CaMKIV and a calcineurin-dependent regulation of nuclear protein phosphatase 1 activity. Prolongation of the synaptic input on the time scale of minutes, in part by an activity-induced inactivation of calcineurin, greatly extends the period over which phospho-CREB levels are elevated, thus affecting induction of downstream genes.

Abstract

Molecular determinants of Ca2+ channel responsiveness to inhibition by receptor-coupled G proteins were investigated in Xenopus oocytes. The inhibitory response of alpha1B (N-type) channels was much larger than alpha1A (P/Q-type) channels, while alpha1C (L-type) channels were unresponsive. Differences in both degree and speed of inhibition were accounted for by variations in inhibitor off-rate. We tested proposals that inhibitory G protein and Ca2+ channel beta subunits compete specifically at the I-II loop. G protein-mediated inhibition remained unaltered in alpha1B subunits containing a point mutation in the I-II loop segment critical for Ca2+ channel beta subunit binding, and in chimeras where the I-II loop of alpha1B was replaced with counterparts from alpha1A or alpha1c. Full interconversion between modulatory behaviors of alpha1B and alpha1A was achieved only by swapping both motif I and the C-terminus in combination. Thus, essential structural elements for G protein modulation reside in multiple Ca2+ channel domains.

Abstract

Hydrogen ions are important regulators of ion flux through voltage-gated Ca2+ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca2+ channels expressed in Xenopus oocytes. WT channels in 150 mM K+ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca2+ interaction. Glu(E)-->Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an approximately 40 pS conductance, the EIIIQ mutant showed the approximately 40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (approximately 145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca2+ channels and provide a simple molecular explanation for the modulatory effect of H+ ions on open channel flux and the competition between H+ ions and permeant divalent cations. The characteristics of H+ interactions advanced our picture of the functional interplay between P-region glutamates, with important implications for the mechanism of Ca2+ selectivity and permeation.

Abstract

It has been established that multiple types of Ca2+ channels participate in triggering neurotransmitter release at central synapses, but there is uncertainty about the nature of their combined actions. We investigated synaptic transmission at CA3-CA1 synapses of rat hippocampal slices and asked whether the dependence on omega-CTx-GVIA-sensitive N-type channels and omega-Aga-IVA-sensitive P/Q-type Ca2+ channels can be altered by physiological mechanisms. The reliance on multiple types of Ca2+ channels was not absolute but depended strongly on the amount of Ca2+ influx through individual channels, which was manipulated by prolonging the presynaptic action potential with the K+ channel blocker 4-aminopyridine (4-AP) and by varying the extracellular Ca2+ concentration ([Ca2+]o). We quantified the influence of spike broadening on Ca2+ influx through various Ca2+ channels by imposing mock action potentials on voltage-clamped cerebellar granule neurons. In field recordings of the EPSP in hippocampal slices, action potential prolongation increased the EPSP slope by 2-fold and decreased its reliance on either N-type or P/Q-type Ca2+ channels. The inhibition of synaptic transmission by N-type channel blockade was virtually eliminated in the presence of 4-AP, but it could be restored by lowering [Ca2+]o. These results rule out a scenario in which a significant fraction of presynaptic terminals rely solely on N-type channels to trigger transmission. The change in sensitivity to the neurotoxins with 4-AP could be explained in terms of a nonlinear relationship between Ca2+ entry and synaptic strength, which rises steeply at low [Ca2+]o, but approaches saturation at high [Ca2+]o. This relationship was evaluated experimentally by varying [CA2+]o in the absence and presence of 4-AP. One consequence of this relationship is that down-modulation of presynaptic Ca2+ channels by various modulators would increase the relative impact of spike broadening greatly.

Abstract

Phosphorylation of the transcription factor CREB is thought to be important in processes underlying long-term memory. It is unclear whether CREB phosphorylation can carry information about the sign of changes in synaptic strength, whether CREB pathways are equally activated in neurons receiving or providing synaptic input, or how synapse-to-nucleus communication is mediated. We found that Ca(2+)-dependent nuclear CREB phosphorylation was rapidly evoked by synaptic stimuli including, but not limited to, those that induced potentiation and depression of synaptic strength. In striking contrast, high frequency action potential firing alone failed to trigger CREB phosphorylation. Activation of a submembranous Ca2+ sensor, just beneath sites of Ca2+ entry, appears critical for triggering nuclear CREB phosphorylation via calmodulin and a Ca2+/calmodulin-dependent protein kinase.

Abstract

Rapid and reliable synaptic transmission depends upon the close proximity of voltage-gated calcium channels and neurotransmitter-containing vesicles in the presynaptic terminal. Although it is clear that a local Ca2+ rise conveys the crucial signal from Ca2+ channels to the exocytotic mechanism, little is known about whether communication ever proceeds in the opposite direction, from the release machinery to Ca2+ channels. To look for such signalling, we examined the interaction of various types of voltage-gated Ca2+ channels with syntaxin, a presynaptic membrane protein of relative molecular mass 35,000 which may play a key part in synaptic vesicle docking and fusion and which interacts strongly with N-type Ca2+ channels. Here we report that co-expression of syntaxin 1A with N-type channels in Xenopus oocytes sharply decreases the availability of these channels. This is due to the stabilization of channel inactivation rather than to a simple block or lack of channel expression, because it is overcome by strong hyperpolarization. Deletion of syntaxin's carboxy-terminal transmembrane domain abolishes its functional effect on Ca2+ channels. Syntaxin produced a similar effect on Q-type Ca2+ channels encoded by alpha 1A but not on L-type Ca2+ channels. Thus, the syntaxin effect is specific for Ca2+ channel types that participate in fast transmitter release in the mammalian central nervous system. We hypothesize that, in addition to acting as a vesicle-docking site, syntaxin may influence presynaptic Ca2+ channels, opposing Ca2+ entry where it is not advantageous, but allowing it at release sites where synaptic vesicles have become docked and/or ready for fusion.

Abstract

Ca2+ channels display remarkable selectivity and permeability, traditionally attributed to multiple, discrete Ca2+ binding sites lining the pore. Each of the four pore-forming segments of Ca2+ channel alpha 1 subunits contains a glutamate residue that contributes to high-affinity Ca2+ interactions. Replacement of all four P-region glutamates with glutamine or alanine abolished micromolar Ca2+ block of monovalent current without revealing any additional independent high-affinity Ca2+ binding site. Pairwise replacements of the four glutamates excluded the hypothesis that they form two independent high-affinity sites. Systematic alterations of side-chain length, charge, and polarity by glutamate replacement with aspartate, glutamine, or alanine weakened the Ca2+ interaction, with considerable asymmetry from one repeat to another. The P-region glutamate in repeat I was unusual in its sensitivity to aspartate replacement but not glutamine substitution. While all four glutamates cooperate in supporting high-affinity interactions with single Ca2+ ions, they also influence the interaction between multiple divalent cations.

Abstract

We have used a focal stimulation method to study neurotransmission at synapses onto hippocampal pyramidal neurons in cultures derived from neonatal rats. Single functional boutons were visualized by activity-dependent preloading with the fluorescent dye FM1-43, then focally stimulated by localized application of elevated K+/Ca2+ solution via a puffer pipette, while postsynaptic currents were recorded under whole cell voltage clamp (Liu and Tsien, 1995). This paper gives a detailed description of the main properties of this experimental system and of information it has provided about fundamental properties of hippocampal synapses. Most of the experiments focused on excitatory postsynaptic currents (EPSCs), but preliminary recordings of inhibitory events (IPSCs) are also reported here. The unitary EPSCs at individual synapses varied greatly in amplitude, but were relatively uniform in their time course. The frequency of the synaptic events was greatly reduced by lowering the external Ca2+ concentration or by application of baclofen, a GABAB receptor agonist. Frequent repetitive stimulation produced a decline in the incidence of EPSCs that was readily reversed upon rest. We attribute the decline to exhaustion of a pool of available vesicles; typically, recovery proceeded with a time constant of approximately 40 sec (23 degrees C), and involved a vesicular pool capable of generating approximately 90 EPSCs without recycling. While synaptic currents were broadly distributed in amplitude (Bekkers et al., 1990), this distribution was remarkably similar at multiple synapses on a given postsynaptic neuron. The median synaptic current amplitude varied 4-fold across different cells, decreasing markedly with increasingly dense synaptic innervation. The implications of these results for cellular signal processing and quantal analysis are discussed.

Abstract

Four different types of Ca2+ channel alpha 1 subunits, representing the major classes of voltage-gated Ca2+ channels, were individually coexpressed along with alpha 2/delta and beta 2b subunits in Xenopus oocytes. These subunits (and the encoded channel types and major tissues of origin) included alpha 1C (L-type, cardiac), alpha 1B (N-type, central nervous system), alpha 1A (P/Q-type, central nervous system), and alpha 1E (most likely R-type, central nervous system). Divalent cation currents through these channels (5 mM Ba2+) were evaluated with the two-microelectrode voltage-clamp technique. The expressed channels were compared with regard to their responses to a structurally novel, nondihydropyridine compound, mibefradil (Ro 40-5967). In the micromolar concentration range, this drug exerted clear inhibitory effects on each of the four channel types, reducing divalent cation current at all test potentials, with the non-L-type channels being more sensitive to inhibition than the L-type channels under fixed experimental conditions. For all channel types, mibefradil was a much more effective inhibitor at more depolarized holding potentials, suggesting tighter binding of the drug to the inactivated state than to the resting state. The difference in apparent affinities of resting and inactivated states of the channels, calculated based on a modulated receptor hypothesis, was 30-70-fold. In addition, the time course of decay of Ca2+ channel current was accelerated in the presence of drug, consistent with open channel block. The effect of increasing stimulation frequency was tested for L-type channels and was found to greatly enhance the degree of inhibition by mibefradil, consistent with promotion of block by channel opening and inactivation. Allowing for state-dependent interactions, the drug concentrations found to block L-, N-, Q-, and R-type channels by 50% are at least 10-fold higher than half-blocking levels previously reported for T-type channels in vascular smooth muscle cells under similar experimental conditions. This may help explain the ability of the drug to spare working myocardium (strongly negative resting potential, dominance of L-type channels in their resting state) while reducing contraction in blood vessels (presumably involving T-type channels or partially inactivated L-type channels). Thus, mibefradil is a new addition to the family of nonselective organic Ca2+ channel inhibitors, as exemplified by bepridil and fluspirilene, and may prove useful as an experimental tool for studying diverse physiological events initiated by Ca2+ influx. It complements classes of drugs with relatively selective effects on L-type channels, as exemplified by nifedipine and diltiazem.

Abstract

Long-term potentiation has previously been studied with electrophysiological techniques that do not readily separate presynaptic and postsynaptic contributions. Changes in exocytotic-endocytotic cycling have now been monitored at synapses between cultured rat hippocampal neurons by measuring the differential uptake of antibodies that recognize the intraluminal domain of the synaptic vesicle protein synaptotagmin. Vesicular cycling increased markedly during glutamate-induced long-term potentiation. The degree of potentiation was heterogeneous, appearing greater at synapses at which the initial extent of vesicular turnover was low. Thus, changes in presynaptic activity were visualized directly and the spatial distribution of potentiation could be determined at the level of single synaptic boutons.

Abstract

Synaptic transmission between individual presynaptic terminals and postsynaptic dendrites is a fundamental element of communication among central nervous system neurons. Yet little is known about evoked neurotransmission at the level of single presynaptic boutons. Here we describe key functional characteristics of individual presynaptic boutons of hippocampal neurons in culture. Excitatory postsynaptic currents (e.p.s.cs) were evoked by localized application of elevated K+/Ca2+ solution to single functional boutons, visually identified by staining with the vital dye FM1-43 (refs 6, 7). Frequent repetitive stimulation produced a decline in the incidence of e.p.s.cs as the pool of releasable vesicles was exhausted; typically, recovery proceeded with a time constant of about 40 s (23 degrees C), and involved a vesicular pool capable of generating about 90 e.p.s.cs without recycling. At individual synapses, synaptic currents were broadly distributed in amplitude, but this distribution was remarkably similar at multiple synapses on a given postsynaptic neuron. The average size of synaptic currents and of responses to focal glutamate application varied fourfold across different cells, decreasing markedly with increasingly dense synaptic innervation. This raises the possibility of a very effective mechanism for coordinating synaptic strength at multiple sites throughout the dendritic tree.

Abstract

The diversity of Ca2+ channel types in rat cerebellar granule neurons was investigated with whole-cell recordings (5 mM external Ba2+). Contributions of five different high-voltage-activated Ca2+ channel current components were distinguished pharmacologically. Nimodipine-sensitive L-type current and omega-CTx-GVIA-sensitive N-type current contributed 15 and 20% of the total current, respectively. The bulk of the remaining current (46%) was inhibited by omega-Aga-IVA. The current blocked by this toxin was further subdivided into two components, P-type and Q-type, on the basis of differences in their inactivation kinetics and sensitivity to omega-Aga-IVA. P-Type current was noninactivating during 0.1 sec depolarizations, half-blocked at about 1-3 nM omega-Aga-IVA, and contributed approximately 11% of the total current; Q-type current was prominently inactivating, half-blocked at approximately 90 nM omega-Aga-IVA, and comprised 35% of the total current. Both P- and Q-type currents were potently inhibited by the Conus magus toxin omega-CTx-MVIIC. A current component resistant to all of the aforementioned blockers (R-type) displayed more rapid inactivation than the other components and constituted 19% of the total current. The Q-type current, the largest of the current components in the granule neurons, resembles currents that can be generated in Xenopus oocytes by expression of cloned alpha 1A subunits.

Abstract

Neurotoxins that selectively block Na+, K+ or Ca2+ channels have provided valuable information about the functional diversity of the voltage-gated channel superfamily. For Ca2+ channels, a variety of toxins have been found to block individual channel types. The best-known example is omega-conotoxin-GVIA, a member of a large family of peptide toxins derived from venomous cone snails, which potently and selectively blocks N-type Ca2+ channels, allowing their purification, cellular localization, and the elucidation of their roles in Ca2+ entry, neurotransmitter release and neuronal migration. In contrast to Na+ and K+ channels, little is known about the molecular features that underlie Ca(2+)-channel susceptibility to toxin block; it is also unknown whether block occurs by direct physical occlusion or an action on channel gating. Here we describe structural determinants of N-type Ca2+ channel's interaction with omega-conotoxin-GVIA. When chimaeras combining individual motifs from the N-type channel and from a channel insensitive to omega-conotoxin-GVIA were expressed in Xenopus oocytes, each of the four motifs appeared to contribute to interaction with the toxin. The most dramatic effects on toxin interactions were seen at a single cluster of residues in the large putative extracellular loop between IIIS5 and IIIH5, consistent with a direct pore-blocking mechanism. These results provide a starting point for delineating the architecture of the outer vestibule of the Ca2+ channel.

Abstract

Voltage-dependent Ca2+ channels respond to membrane depolarization by conformational changes that control channel opening and eventual closing by inactivation. The kinetics of inactivation differ considerably between types of Ca2+ channels and are important in determining the amount of Ca2+ entry during electrical activity and its resulting impact on diverse cellular events. The most intensively characterized forms of inactivation in potassium and sodium channels involve pore block by a tethered plug. In contrast, little is known about the molecular basis of Ca(2+)-channel inactivation. We studied the molecular mechanism of inactivation of voltage-gated calcium channels by making chimaeras from channels with different inactivation rates. We report here that the amino acids responsible for the kinetic differences are localized to membrane-spanning segment S6 of the first repeat of the alpha 1 subunit (IS6), and to putative extracellular and cytoplasmic domains flanking IS6. Involvement of this region in Ca(2+)-channel inactivation was unexpected and raises interesting comparisons with Na+ channels, where the III-IV loop is a critical structural determinant. Ca(2+)-channel inactivation has some features that resemble C-type inactivation of potassium channels.

Abstract

The presence of nitric oxide synthase (NOS) in CA1 pyramidal cells of the rat hippocampus was demonstrated by single-cell PCR. NOS-specific primers were used to amplify mRNA isolated from single hippocampal neurons. The sequence of the major amplification-product obtained was identical to that of the constitutively expressed brain-isoform of NOS. These results confirm immunocytochemical data that NOS is present in CA1, and, therefore, nitric oxide could function as a retrograde messenger in long-term potentiation.

Abstract

This study describes a Ca2+ store in fura-2-loaded bullfrog sympathetic neurons that modulates [Ca2+]i responses elicited by either depolarization or Ca2+ release from a caffeine- and ryanodine-sensitive store. This store is insensitive to caffeine and ryanodine, but is sensitive to the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The FCCP-sensitive store slows both the rise in [Ca2+]i during stimulation (apparently by accumulating Ca2+ from the cytosol) and the recovery following stimulation (by releasing the accumulated Ca2+ into the cytosol). For a fixed level of depolarization, recovery is slowed to an extent that depends on stimulus duration. [Ca2+]i imaging shows that these effects are prominent in the soma but not in growth cones. Ca2+ uptake by the FCCP-sensitive store appears to be strongly [Ca2+]i dependent, since it becomes influential only when [Ca2+]i approaches approximately 500 nM. Therefore, this store may specifically influence [Ca2+]i during moderate and strong stimulation. The effect of the store on responses to depolarization can be accounted for by a simple three-compartment scheme consisting of the extracellular medium, the cytosol, and a single internal store with a [Ca2+]i-dependent uptake mechanism resembling the mitochondrial Ca2+ uniporter. The store's effect on responses to caffeine-induced Ca2+ release can be accounted for by including a second internal compartment to represent the caffeine-sensitive store. While the identity of the FCCP-sensitive store is unknown, its sensitivity to FCCP is consistent with a mitochondrial pool. It is suggested that by modulating the temporal properties of [Ca2+]i following stimulation, the FCCP-sensitive store may influence the degree of activation of intracellular [Ca2+]i-dependent processes.

Abstract

There has been rapid progress in understanding the structural basis of ion selectivity and permeation in both ligand- and voltage-gated channels. Recognition of similarities in overall architecture within a channel class has led to an increasing focus on the specific molecular determinants that endow a channel with its own distinctive character. It has been possible in some cases to identify individual amino acids essential for ion selectivity.

Abstract

Several types of calcium channels found in the central nervous system are possible participants in triggering neurotransmitter release. Synaptic transmission between hippocampal CA3 and CA1 neurons was mediated by N-type calcium channels, together with calcium channels whose pharmacology differs from that of L- and P-type channels but resembles that of the Q-type channel encoded by the alpha 1A subunit gene. Blockade of either population of channels strongly increased enhancement of synaptic transmission with repetitive stimuli. Even after complete blockade of N-type channels, transmission was strongly modulated by stimulation of neurotransmitter receptors or protein kinase C. These findings suggest a role for alpha 1A subunits in synaptic transmission and support the idea that neurotransmitter release may depend on multiple types of calcium channels under physiological conditions.

Abstract

It has been proposed that nitric oxide (NO) serves as a key retrograde messenger during long-term potentiation at hippocampal synapses, linking induction of long-term potentiation in postsynaptic CA1 pyramidal cells to expression of long-term potentiation in presynaptic nerve terminals. However, nitric oxide synthase (NOS), the proposed NO-generating enzyme, has not yet been detected in the appropriate postsynaptic cells. We here demonstrate specific NOS immunoreactivity in the CA1 region of hippocampal sections by using an antibody specific for NOS type I and relatively gentle methods of fixation. NOS immunoreactivity was found in dendrites and cell bodies of CA1 pyramidal neurons. Cultured hippocampal pyramidal cells also displayed specific immunostaining. Control experiments showed no staining with preimmune serum or immune serum that was blocked with purified NOS. These results demonstrate that CA1 pyramidal cells contain NOS, as required were NO involved in retrograde signaling during hippocampal synaptic plasticity.

Abstract

Voltage-gated Ca2+ channels link changes in membrane potential to the delivery of Ca2+, a key second messenger for many cellular responses. Ca2+ channels show selectivity for Ca2+ over more plentiful ions such as Na+ or K+ by virtue of their high-affinity binding of Ca2+ within the pore. It has been suggested that this binding involves four conserved glutamate residues in equivalent positions in the putative pore-lining regions of repeats I-IV in the Ca2+ channel a1 subunit. We have carried out a systematic series of single amino-acid substitutions in each of these positions and find that all four glutamates participate in high-affinity binding of Ca2+ or Cd2+. Each glutamate carboxylate makes a distinct contribution to ion binding, with the carboxylate in repeat III having the strongest effect. Some single glutamate-to-lysine mutations completely abolish micromolar Ca2+ block, indicating that the pore does not possess any high-affinity binding site that acts independently of the four glutamate residues. The prevailing model of Ca2+ permeation must thus be modified to allow binding of two Ca2+ ions in close proximity, within the sphere of influence of the four glutamates. The functional inequality of the glutamates may be advantageous in allowing simultaneous interactions with multiple Ca2+ ions moving single-file within the pore. Competition among Ca2+ ions for individual glutamates, together with repulsive ion-ion electrostatic interaction, may help achieve rapid flux rates through the channel.

Abstract

We used the fluorescent membrane probe FM 1-43 to label recycling synaptic vesicles within the presynaptic boutons of dissociated hippocampal neurons in culture. Quantitative time-lapse fluorescence imaging was employed in combination with rapid superfusion techniques to study the dynamics of synaptic vesicles within single boutons. This approach enabled us to measure exocytosis and to analyze the kinetics of endocytosis and the preparation of endocytosed vesicles for re-release (repriming). Our measurements indicate that under sustained membrane depolarization, endocytosis persists much longer than exocytosis, with a t1/2 approximately 60 s (approximately 24 degrees C); once internalized, vesicles become reavailable for exocytosis in approximately 30 s. Furthermore, we have shown that endocytosis is not dependent on membrane potential and, unlike exocytosis, that it is independent of extracellular Ca2+.

Abstract

Transcripts for the class A Ca2+ channel alpha 1 subunit (also known as BI) are present at high levels in many parts of the mammalian CNS and are widely assumed to encode the P-type Ca2+ channel. To characterize the biophysical and pharmacological properties of alpha 1A channels, macroscopic and single-channel recordings were made in Xenopus oocytes injected with alpha 1A cRNA. alpha 1-specific properties were identified by making systematic comparisons with the more familiar class C alpha 1 subunit under the condition of a standard ancillary subunit (alpha 2/delta + beta) makeup. alpha 1A currents activate and inactivate more rapidly and display steeper voltage dependence of gating than alpha 1C currents. Unlike alpha 1C, alpha 1A channels are largely insensitive to dihydropyridines and FPL 64176, but respond to the cone snail peptide omega-CTx-MVIIC(SNX-230), a potent and fairly selective inhibitor. In comparison with P-type Ca2+ channels in rat cerebellar Purkinje cells, alpha 1A channels in oocytes are approximately 10(2)-fold less sensitive to omega-Aga-IVA and approximately 10-fold more sensitive to omega-CTx-MVIIC. alpha 1A channels are not inhibited by Bay K 8644 and inactivate much more rapidly than P-type Ca2+ channels. Thus, alpha 1A is capable of generating a Ca2+ channel phenotype quite different from P-type current.

Abstract

Diverse types of calcium channels in vertebrate neurons are important in linking electrical activity to transmitter release, gene expression and modulation of membrane excitability. Four classes of Ca2+ channels (T, N, L and P-type) have been distinguished on the basis of their electrophysiological and pharmacological properties. Most of the recently cloned Ca2+ channels fit within this functional classification. But one major branch of the Ca2+ channel gene family, including BII (ref. 15) and doe-1 (ref. 16), has not been functionally characterized. We report here the expression of doe-1 and show that it is a high-voltage-activated (HVA) Ca2+ channel that inactivates more rapidly than previously expressed calcium channels. Unlike L-type or P-type channels, doe-1 is not blocked by dihydropyridine antagonists or the peptide toxin omega-Aga-IVA, respectively. In contrast to a previously cloned N-type channel, doe-1 block by omega-CTx-GVIA requires micromolar toxin and is readily reversible. Unlike most HVA channels, doe-1 also shows unusual sensitivity to block by Ni2+. Thus, doe-1 is an HVA Ca2+ channel with novel functional properties. We have identified a Ca2+ channel current in rat cerebellar granule neurons that resembles doe-1 in many kinetic and pharmacological features.

Abstract

In many neurons, transmitter release from presynaptic terminals is triggered by Ca2+ entry via dihydropyridine-insensitive Ca2+ channels. We have looked for cDNAs for such channels in the nervous system of the marine ray Discopyge ommata. One cDNA (doe-2) is similar to dihydropyridine-sensitive L-type channels, and two cDNAs (doe-1 and doe-4) are similar to the subfamily of dihydropyridine-insensitive non-L-type channels. doe-4, which encodes a protein of 2326 aa, most closely resembles a previously cloned N-type channel. doe-1, which encodes a protein of 2223 aa, is a member of a separate branch of the non-L-type channels. Northern blot analysis reveals that doe-1 is abundant in the forebrain. doe-4 is more plentiful in the electric lobe and, therefore, may control neurotransmitter release in motor nerve terminals. These results show that the familial pattern of Ca(2+)-channel genes has been preserved from a stage in evolution before the divergence of higher and lower vertebrates > 400 million years ago. The cloning of these channels may be a useful starting point for elucidating the role of the Ca2+ channels in excitation-secretion coupling in nerve terminals.

Abstract

G protein-mediated inhibition of voltage-activated calcium channels by neurotransmitters has important consequences for the control of synaptic strength. Single-channel recordings of N-type calcium channels in frog sympathetic neurons reveal at least three distinct patterns of gating, designated low-Po, medium-Po, and high-Po modes according to their probability of being open (Po) at -10 millivolts. The high-Po mode is responsible for the bulk of divalent cation entry in the absence of neurotransmitter. Norepinephrine greatly decreased the prevalence of high-Po gating and increased the proportion of time a channel exhibited low-Po behavior or no activity at all, which thereby reduced the overall current. Directly observed patterns of transition between the various modes suggest that activated G protein alters the balance between modal behaviors that freely interconvert even in the absence of modulatory signaling.

Abstract

The effect of protein kinase C (PKC) stimulation on Ca2+ channels was studied in frog sympathetic neurons. 12,13-Phorbol dibutyrate (PDBu) consistently augmented Ca2+ channel currents in whole-cell recordings. This enhancement was blocked by staurosporine and PKC(19-31), but not produced by 4 alpha-phorbol 12,13-didecanoate, indicating that PDBu acts via PKC. Both N- and L-type currents, as isolated pharmacologically, were increased. PKC enhancement was independent of the extent of G protein activation, indicating that it was not caused by removal of tonic G protein inhibition. In unitary recordings PDBu produced dramatic increases in single N- and L-type channel activity by sharply decreasing closed time intervals between adjacent openings, but did not alter the unitary current size or mean open time. This up-modulation by PKC may constitute a positive feedback mechanism in the regulation of neuronal Ca2+ channel activity.

Abstract

In many neurons, N-type Ca2+ channels are a major Ca2+ entry pathway and strongly influence neurotransmitter release. We carried out cell-attached patch recordings (110 mM Ba2+ as charge carrier) to characterize the rapid opening and closing kinetics of N-type Ca2+ channel gating in frog sympathetic neurons. Single channels display at least three distinct patterns of gating, characterized as low-, medium-, and high-rho o modes on the basis of channel open probability (rho o) during depolarizing pulses to -10 mV. Spontaneous transitions from one mode to another are infrequent, with an exponential distribution of dwell times and mean sojourns of approximately 10 sec in each mode. Thus, a channel typically undergoes hundreds or thousands of open-closed transitions in one mode before switching to a different mode. Transitions between modes during a depolarization were occasionally detected, but were rare, as expected for infrequent modal switching. Within each mode, the activation kinetics were well described by a simple scheme (C2-C1-O), as previously reported for other types of Ca2+ channels. Rate constants are strikingly different from one mode to another, giving each mode its own characteristic kinetic signature. The gating behavior at -10 mV ranges from brief openings (approximately 0.3 msec) and long closures (10-20 msec) for low-rho o gating to long openings (3 msec) and brief closures (approximately 1 msec) for high-rho o gating. Intermediate values for mean open durations (approximately 1.5 msec) and mean closed durations (approximately 3 msec) were found for medium-rho o gating. In addition to being kinetically distinct, channel openings in the low-rho o mode often exhibit a unitary current approximately 0.2 pA larger than in the medium- or high-rho o mode. Each mode is characterized by its own voltage dependence: activation occurs at relatively negative potentials and is most steeply voltage dependent in the high-rho o mode, while activation requires very strong depolarizations and is weakly voltage dependent in the low-rho o mode. The proportion of time spent in the individual modes varies greatly from one patch to another, suggesting that modal gating may be subject to cellular control.

Abstract

Sympathetic neurons display robust [Ca2+]i oscillations in response to caffeine and mild depolarization. Oscillations occur at constant membrane potential, ruling out voltage-dependent changes in plasma membrane conductance. They are terminated by ryanodine, implicating Ca(2+)-induced Ca2+ release. Ca2+ entry is necessary for sustained oscillatory activity, but its importance varies within the oscillatory cycle: the slow interspike rise in [Ca2+]i requires Ca2+ entry, but the rapid upstroke does not, indicating that it reflects internal Ca2+ release. Sudden alterations in [Ca2+]o, [K+]o, or [caffeine]o produce immediate changes in d[Ca2+]i/dt and provide information about the relative rates of surface membrane Ca2+ transport as well as uptake and release by internal stores. Based on our results, [Ca2+]i oscillations can be explained in terms of coordinated changes in Ca2+ fluxes across surface and store membranes.

Abstract

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.

Abstract

1. We studied how in changes in cytosolic free Ca2+ concentration ([Ca2+]i) produced by voltage-dependent Ca2+ entry are influenced by a caffeine-sensitive Ca2+ store in bullfrog sympathetic neurones. Ca2+ influx was elicited by K+ depolarization and the store was manipulated with either caffeine or ryanodine. 2. For a time after discharging the store with caffeine and switching to a caffeine-free medium: (a) [Ca2+]i was depressed by up to 40-50 nM below the resting level, (b) caffeine responsiveness was diminished, and (c) brief K+ applications elicited [Ca2+]i responses with slower onset and faster recovery than controls. These effects were more pronounced as the conditioning caffeine concentration was increased over the range 1-30 mM. 3. [Ca2+]i, caffeine and K+ responsiveness recovered in parallel with a half-time of approximately 2 min. Recovery required external Ca2+ and was speeded by increasing the availability of cytosolic Ca2+, suggesting that it reflected replenishment of the store at the expense of cytosolic Ca2+. 4. During recovery, Ca2+ entry stimulated by depolarization had the least effect on [Ca2+]i when the store was filling most rapidly. This suggests that the effect of Ca2+ entry on [Ca2+]i is modified, at least in part, because some of the Ca2+ which enters the cytosol during stimulation is taken up by the store as it refills. 5. Further experiments were carried out to investigate whether the store can also release Ca2+ in response to stimulated Ca2+ entry. In the continued presence of caffeine at a low concentration (1 mM), high K+ elicited a faster and larger [Ca2+]i response compared to controls; at higher concentrations of caffeine (10 and 30 mM) responses were depressed. 6. Ryanodine (1 microM) reduced the rate at which [Ca2+]i increased with Ca2+ entry, but not to the degree observed after discharging the store. At this concentration, ryanodine completely blocked responses to caffeine but had no detectable effect on Ca2+ channel current or the steady [Ca2+]i level achieved during depolarization. 7. We propose that, depending on its Ca2+ content, the caffeine-sensitive store can either attenuate or potentiate responses to depolarization. When depleted and in the process of refilling, the store reduces the impact of Ca2+ entry as some of the Ca2+ entering the cytosol during stimulation is captured by the store.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract

Long-term potentiation (LTP) is an example of a persistent change in synaptic function in the mammalian brain, thought to be essential for learning and memory. At the synapse between hippocampal CA3 and CA1 neurons LTP is induced by a Ca2+ influx through glutamate receptors of the NMDA (N-methyl-D-aspartate) type (see Collingridge et al 1992, this volume). How does a rise in [Ca2+]i lead to enhancement of synaptic function? We have tested the popular hypothesis that Ca2+ acts via a Ca(2+)-dependent protein kinase. We found that long-lasting synaptic enhancement was prevented by prior intracellular injection of potent and selective inhibitory peptide blockers of either protein kinase C (PKC) or Ca2+/calmodulin-dependent protein kinase II (CaMKII), such as PKC(19-31) or CaMKII(273-302), but not by control peptides. Evidently, activity of both PKC and CaMKII is somehow necessary for the postsynaptic induction of LTP. To determine if these kinases are also involved in the expression of LTP, we impaled cells with microelectrodes containing protein kinase inhibitors after LTP had already been induced. Strikingly, established LTP was not suppressed by a combination of PKC and CaMKII blocking peptides, or by intracellular postsynaptic H-7. However, established LTP remained sensitive to bath application of H-7. Thus, the persistent signal may be a persistent kinase, but if so, the kinase cannot be accessed within the postsynaptic cell. Evidence for a presynaptic locus of expression comes from our studies of quantal synaptic transmission under whole-cell voltage clamp. We find changes in synaptic variability expected to result from enhanced presynaptic transmitter release, but little or no increase in quantal size. Furthermore, miniature synaptic currents in hippocampal cultures are increased in frequency but not amplitude as a result of a glutamate-driven postsynaptic induction. The combination of postsynaptic induction and presynaptic expression necessitates a retrograde signal from the postsynaptic cell to the presynaptic terminal.

Abstract

Voltage-dependent Ca2+ channels regulate Ca2+ entry and thereby contribute to Ca2+ signalling in many cells. Functional studies have uncovered several types of Ca2+ channel, distinguished by pharmacology, electrophysiology and tissue localization. More recently, molecular cloning has revealed an even greater diversity among Ca2+ channels, arising from multiple genes and alternative splicing. L-type, dihydropyridine-sensitive Ca2+ channels have been the most extensively characterized to date. Recently, Numa's group has reported the cloning and expression of a dihydropyridine-insensitive Ca2+ channel from brain that most closely resembles the P-type channel described by Llinas and colleagues. These results contribute to rapidly growing knowledge about molecular determinants of Ca2+ channel diversity.

Abstract

A peptide toxin from a Conus marine snail, omega-conotoxin GVIA (omega-CgTx) has been used extensively as a probe for certain types of neuronal calcium channels. It is often assumed that omega-CgTx interacts with Ca2+ channels exclusively. We have tested this assumption in a study of omega-CgTx-binding sites in the electric organ of Discopyge ommata. Synaptosomal membranes from this tissue contain low affinity omega-CgTx receptor sites (Kd = 0.6 microM) in great abundance (280 pmol/mg of protein), as first reported by Ahmad and Miljanich (Ahmad, S. N., and Miljanich, G.P. (1988) Brain Res. 453, 247-256). However, we find that a large majority of these omega-CgTx-binding sites co-purify with the nicotinic acetylcholine receptor (nAChR) and can be immunoprecipitated by monoclonal antibodies generated against the nAChR of Torpedo. Cross-linking experiments with radiolabeled omega-CgTx show pronounced specific labeling of the alpha-subunit of the nAChR but not other subunits. Specific omega-CgTx binding to the nAChR is reduced by millimolar Ca2+ but not by alpha- or kappa-bungarotoxin, alpha-conotoxin, or carbamylcholine. Cross-linking experiments also reveal omega-CgTx-binding proteins of 170 and 60 kDa. The characteristics of the 170-kDa protein make it a likely candidate for the alpha 1-subunit of an N-type Ca2+ channel.

Abstract

Influx of Ca2+ through NMDA channels may initiate the stabilization of coactive synapses during development of the retinotectal projection in frogs. Ca2+ imaging techniques were applied to cultured tectal cells to investigate whether excitatory amino acids cause a rise in [Ca2+]i. High [K+], NMDA, and glutamate increase [Ca2+]i in about 75% of the cells. NMDA and glutamate responses were completely blocked in the absence of extracellular Ca2+ and by the NMDA receptor or channel blockers APV and MK-801. The NMDA response was also blocked by Mg2+. Quisqualate and kainate produced little or no rise in [Ca2+]i. These studies indicate that when tectal cells are exposed to the retinal ganglion cell transmitter glutamate, the predominant means of Ca2+ entry is through NMDA channels.

Abstract

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a widely studied model system for understanding the cellular mechanisms of memory. In region CA1, LTP is triggered postsynaptically by Ca2(+)-dependent activation of protein kinases, but the locus of persistent modification remains controversial. Statistical analysis of synaptic variability has been proposed as a means of settling this debate, although a major obstacle has been the poor signal-to-noise ratio of conventional intracellular recordings. We have applied the whole-cell voltage clamp technique to study synaptic transmission in conventional hippocampal slices (compare refs 28-30). Here we report that robust LTP can be recorded with much improved signal resolution and biochemical access to the postsynaptic cell. Prolonged dialysis of the postsynaptic cell blocks the triggering of LTP, with no effect on expression of LTP. The improved signal resolution unmasks a large trial-to-trial variability, reflecting the probabilistic nature of transmitter release. Changes in the synaptic variability, and a decrease in the proportion of synaptic failures during LTP, suggest that transmitter release is significantly enhanced.

Abstract

Neuropeptides are known to modulate the excitability of frog sympathetic neurons by inhibiting the M-current and increasing the leak current, but their effects on Ca2+ channels are poorly understood. We compared effects of LHRH, substance P, epinephrine, and muscarine on Ca2+, K+, and leak currents in dissociated frog sympathetic neurons. At concentrations that inhibit M-current, LHRH and substance P strongly reduced N-type Ca2+ current and induced a leak conductance that may contribute to slow EPSPs. In contrast, muscarine produced little reduction of Ca2+ current, even in cells in which it strongly suppressed the M-current. We find that peptidergic inhibition of Ca2+ channels involves G proteins, but does not require protein kinases. In addition, it leads to reductions in Ca2(+)-activated K+ current and catecholamine release.

Abstract

Long-term potentiation (LTP) of synaptic transmission is a widely studied cellular example of synaptic plasticity. However, the identity, localization, and interplay among the biochemical signals underlying LTP remain unclear. Intracellular microelectrodes have been used to record synaptic potentials and deliver protein kinase inhibitors to postsynaptic CA1 pyramidal cells. Induction of LTP is blocked by intracellular delivery of H-7, a general protein kinase inhibitor, or PKC(19-31), a selective protein kinase C (PKC) inhibitor, or CaMKII(273-302), a selective inhibitor of the multifunctional Ca2+-calmodulin-dependent protein kinase (CaMKII). After its establishment, LTP appears unresponsive to postsynaptic H-7, although it remains sensitive to externally applied H-7. Thus both postsynaptic PKC and CaMKII are required for the induction of LTP and a presynaptic protein kinase appears to be necessary for the expression of LTP.